1. Field of the Invention
The present invention relates to low-photon flux image-intensified electronic cameras, and in particular to ones that use gallium arsenide phosphide photocathodes and intensifier tubes chilled below zero degrees centigrade with a dual microchannel plate structure, and without an ion barrier film.
2. Description of the Prior Art
Bioluminescent living tissues can be engineered for use in medical studies of live animals, plant cells, plants, and vitro biological samples. A good background in this area was published in the Journal of Biomedical Optics 6(4), 432–440 (October 2001), by B. W. Rice, et al., in an article titled, “In vivo imaging of light-emitting probes.”
If the bioluminescent tissues are on the surface of an organism, the light emitted can be relatively easy to image with a camera. But if the bioluminescent tissues are internal organs or other structures like tumors, the intervening tissues can reduce the light reaching the camera to levels that can require exposure times well in excess of several minutes just to detect an image.
Luciferase is a photoactive reporter gene that can be imaged in living organisms. Such has been used in laboratory animals and specimens to assess the progression of angiogenesis over time. Transgenic mice used in such research carry a luciferase reporter and a human vascular endothelial growth factor. Living organisms can emit visible light when a luciferin substrate is catalyzed by luciferase and reacts with molecular oxygen. The resulting bioluminescent light is green to red, appears as a result of a chemiluminescent reaction that requires none of the optical excitation needed for fluorescence.
In-vivo imaging can be used to track the progression of a pathogen or tumor in a specimen. The pathogen or tumor is made visible for imaging by modifying the cells to bioluminescence. It is very desirable to be able to superimpose an image of the bioluminescent light on the image of the specimen, e.g., in order to locate and assess the pathogen or tumor relative to its host. But the two cannot be simultaneously shuttered because of the vast difference in light levels.
With prior art charge-coupled bioluminescence camera systems, a five minute long-exposure of an animal subject is used to generate a digital image by collecting a sufficient number of photons in each pixel to generate an image signal that exceeds the image sensor's noise-floor. Imaged over time, the mice in one study showed an increase in bioluminescence that indicated an expression of human vascular endothelial growth factor.
In their article titled, “Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent Escherichia coli in the Neutropenic Mouse Thigh Model of Infection”, Antimicrobial Agents and Chemotherapy, January 2001, p. 129–137, Vol. 45, No. 1, H. L. Rocchetta, et al., reported that a noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopyplasmid. Such bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection.
Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin). A strong correlation existed between bioluminescence levels and viable cell counts. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was reported to be a sound indicator of therapeutic success. Dose-dependent responses were also detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. By monitoring bioluminescence within live animals, these researchers were able to compare the virulence of three strains of Salmonella enterica serovar Typhimurium, which carried the lux genes of P. luminescens on a multicopy plasmid. In addition, orally infected animals treated with the antibiotic ciprofloxacin were shown to have reduced bioluminescence over the abdominal area.
The ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy.
Very low light levels from such tissues can be electronically obscured by the background noise or dark currents thermally generated by camera image devices. Prior-art intensified cameras needed long imaging times and suffer from spurious noise events, high dark counts, high integrated background levels that build with long exposures, and high amplitude “scintillation” ion-feedback noise.
Conventional bi-alkali material photocathodes used in intensified platforms have low quantum efficiencies, high background noise, poor resolution and cosmetic quality, and are typically lens-coupled to a charge-coupled device (CCD). Lens-coupling is relatively inefficient and reduces light-collection efficiencies. Higher gains are therefore needed, and higher gains make the whole more susceptible to scintillation and cosmic ray artifacts in the images.
Chilling has been used to reduce thermally generated noise in electronic devices, but sometimes the amount of cooling needed is extraordinary, expensive, and impractical. Cooling the CCD as low as −90° C. is required to reduce dark current in these devices, and back-thinning is used to improve quantum efficiency. Cooled CCD cameras are reported to have reduced read noise levels of 3–5 electrons, and this limits the detection threshold to 10–20 photons per pixel per sample collection interval, for example.
Olympus Biosystems (Germany) has an Internet website at http://www.olympus-biosystems.com, that explains intensified CCD (ICCD) cameras are basically full-performance CCD cameras optically coupled in two possible ways to an intensifier. A so-called proximity-focused intensifier or wafer tube comprises an entrance window, a photocathode, a microchannel plate (MCP) electron multiplier, and a phosphorescent output screen. The photocathode converts the photons into electrons via the photoelectric effect. The quantum efficiency of the conversion is an important parameter and depends on the coating material which differs in the different generations of intensifiers. The photoelectrons are driven to the MCP which is set under a field of several hundred volts. The MCP contains millions of parallel channels with a diameter of about six micrometers in the newest generations. The channels are coated with a secondary electron emitter which generates more electrons when hit by passing electrons. The intensification gain caused by the avalanche effect of multiple collisions is adjustable over a wide range up to several 10,000. The electrons are accelerated by a voltage of several kilovolts upon exiting the MCP before reaching the phosphor screen. They are converted back into photons with an additional multiplication factor. Conventionally, the screen output light is then relayed to the CCD chip either by a lens or fiber-optic coupling. The advantage of relay lens coupling is the possibility of constructing removable intensifiers that enable to easily convert the ICCD camera reversibly into a standard CCD camera or retrofit an existing camera. However, the light efficiency is a function of transmission and inversely of the square of magnification and lens f-number. It is limited and causes a significant loss of signal and a reduced signal-to-noise ratio.
According to Olympus Biosystems, a much more efficient method to optically couple intensifier and CCD chip is with a fiber-optic taper. However, such component requires a very sophisticated manufacturing process. A fiberoptic taper is a bundle of microscopic fibers 2–3 microns in diameter that guide light from the fluorescent screen to the CCD chip. There are up to nine fibers per pixel usually machined directly onto the diode array. Each microfiber has a cladding to maximize light transmission and a stray-light absorbing coating to contain leakage and prevent the resulting contrast reduction. The signal-to-noise ratio of prior art ICCD cameras is usually worse than that of simple cooled and back-thinned CCD cameras due to the inclusion of several additional noise sources in the intensification stage, e.g., thermal noise from the photocathode, multiplication noise from the MCP, and ion-feedback scintillation noise.